Since the Cretaceous Period, animals have undergone great speciation and divergence. The types of viruses infecting these evolving animal species must also have been subjected to evolutionary pressures resulting in the selection of viruses that now exist. Recently, it has been found that certain leukemias in animals are associated with certain retroviruses. Though it has not been conclusively shown, it is believed that the various leukemias are caused by those retroviruses. Among the leukemia-associated viruses now known are the murine leukemia-associated viruses Murine Leukemia Virus (MuLV), Mink Cell Focus-Forming Virus (MCF) and AKR Ecotropic Virus (Akv); feline leukemia-associated viruses such as Feline Leukemia Virus (FeLV); and human leukemia-associated viruses Human T-Cell Leukemia Virus (HTLV) and Adult T-Cell Leukemia Virus (ATLV). It has also recently been shown that ATLV and HTLV are closely related.
The leukemia-associated viruses are C-type retroviruses having their genetic information stored on RNA encased in a core which in turn is surrounded by an envelope. The strand of RNA has three genes arranged in the following order from the 5' to the 3' end: gag, pol, and env. The gag gene encodes for a nonglycosylated precursor molecule that is proteolytically processed into smaller proteins that are used to construct the core. The pol gene encodes for a protein that is proteolytically processed to form a RNA-dependent DNA polymerase (reverse transcriptase) necessary for translating the RNA virus gene into DNA for use by the cell.
The third gene, env, is adjacent a long terminal repeat sequence (LTR) and encodes for a polyprotein. This polyprotein is glycosylated and proteolytically processed into two smaller proteins of unequal size, the longer protein being linked by a disulfide bond to the smaller protein.
In the case of FeLV, these two envelope proteins have weights of 70 and 15 kilodaltons and are known respectively as gp70 and p15E. The larger envelope protein (e.g., gp70) is amino-terminus oriented while the smaller protein (e.g., p15E) is carboxy-terminus oriented on the polyprotein. These proteins together with a lipid membrane form the envelope protecting the virus core. The large glycosylated protein forms a knob-like structure linked by the disulfide bond to the small protein that forms a spike-like structure extending through the lipid membrane.
Exemplary of leukemia-associated viruses is the Feline Leukemia Virus (FeLV). Infections by FeLV have been extensively studied and serve as a useful model for leukemia-associated viruses. FeLV is horizontally transferred, that is directly from cat to cat, predominantly by contact with salivary and nasal secretions through eating utensils and mutual grooming.
Within six weeks of infection with FeLV, the cat develops usually one of two major host-virus relationships. The first is a self-limiting infection (regressive infection) in which the cat develops sufficient antibodies against both the Feline Leukemia virus and lymphomas which express the feline oncornavirus-associated cell membrane antigen (FOCMA). These cats do not develop FeLV-related diseases and a short time after infection neither FeLV nor FeLV antigens can be detected in the cat's tissue.
The second major host-virus relationship is the persistant active infection (progressive infection) which generally leads to the death of the cat. Death can be caused by proliferative disorders such as lymphosarcoma and leukemia, and anti-proliferative diseases such as aplastic anemia, and secondary viral or bacterial infections that arise because of the immunosuppression caused by FeLV. See generally Feline Leukemia, by Richard G. Olsen, CRC Press, USA. (1981).
FeLV is infectious and replicates freely in cats cells (exogenous) and has been identified as having three specific serotypes or subgroups, which have been designated as A, B, and C, based on the presence of specific virion envelope antigens. Of the three types of FeLV, serotype A designated as FeLV-A, is the most commonly isolated subgroup. FeLV-B is generally found in association with FeLV-A, and FeLV-C is relatively rare and has been isolated along with FeLV-A and FeLV-B from cats with polycythemia. Nucleic acid hybridization studies show structural homologies of at least 85 percent between the three subgroups while greater divergence is noted by Tl oligonucleotide analysis.
There have been previous attempts to prepare vaccines against Feline Leukemia virus. Unfortunately, while some of these vaccines have produced antibodies against FeLV, many not only failed to protect cats from challenge by FeLV, but when the cats were challenged, there was earlier death and larger mean tumor size in comparison with control cats that were not vaccinated. See Salerno, et al. Proc. Soc. Exp. Biol. Med., 160, 18 (1979) and Nathes et al., Cancer Res., 39, 950 (1979).
It would be desirable to produce a vaccine and antibodies that would provide protection against a leukemia-associated virus and the diseases it causes. Historically, vaccines and antibodies have been prepared by killing or attenuating viruses and then injecting the resulting virus particles into a patient or host animal. However, such vaccines always have the inherent threat that the virus may not be completely killed or sufficiently attenuated. The "vaccine" sometimes itself causes disease.
The threat of unattenuated viruses can sometimes be overcome by using only a portion of the virus. This portion is usually a protein from a capsid or envelope which forms the outer portion of the virus. However, even this method is not without well-known difficulties including possible pathogenic responses. The produced vaccine may include antigens that compete with or are even detrimental to the desired immune response. Other antigenic material may also be present that is unrelated to the desired immune response and can cause undesirable side effects.
Various attempts have been made to manufacture vaccines and antibodies to other diseases by other methods. These methods include producing antigen and antibody-producing cells by recombinant DNA techniques and hybridoma methods. However, these methods in addition to being relatively complicated and expensive, are time consuming and have relatively low yields both quantitatively and qualitatively. Great care must be taken in preparing the vaccine or antibody producing cell and in harvesting the desired product. There is also concern about the safety and reliability of any method that requires the desired product be separated from undesired, possibly pathogenic components.
Recently, certain pathogen-related proteins have been immunologically mimicked by a synthetic polypeptide whose sequence corresponds to that of a determinant domain of the pathogen-related protein. Such findings are reported by Sutcliffe et al., Nature, 287, 801-805 (1980); Lerner et al., Proc. Natl. Acad. Sci. USA, 78, 340-347 (1981); and Bittle et al., Nature, 298, 30-33 (1982). A review of the subject was reported by Sutcliffe et al., Science, 219:660-666 (1983). The peptide sequence of a natural protein can be determined from the protein itself or from the nucleotide sequence of the genome encoding that protein.
While the general concept of preparing synthetic antigens has been described, there remains a large area that continues to defy predictability. The field remains largely a matter of speculation, and of trial and error. The many steps needed to determine a retrovirus protein sequence from the RNA genome make it far from certain that any peptide sequence derived will have the desired immunogenic properties.
For example, the enzyme reverse transcriptase is used to polymerize nucleotides into a strand of DNA complementary to the viral RNA genome. Since about one nucleotide in five hundred is miscopied, the resulting DNA can be in error. In addition, the virus RNA molecule used to make the DNA can itself contain errors in transcription. The virus RNA was made in a living cell by RNA polymerase from DNA which in turn was made by reverse transcriptase in a similar process.
After the DNA copy has been prepared, it is then linked to a plasmid vector, a process which has been shown to often cause rearrangements in the cloned DNA fragment. The plasmid is then introduced into a bacterium, and transformed bacterial colonies carrying the recombinant plasmid are selected. A transformed colony is fragmented by streaking on a growth plate and a single isolate is picked for large scale growth and DNA preparation. Since many rounds of DNA replication have occurred in this process, one or more nucleotide-altering events can alter the gene of interest. There is no selective pressure for the bacterium to maintain the desired virus gene. A change could render the DNA sequence of such a gene meaningless or greatly lessen its utility as a blueprint for peptide sequence selection. There is no guarentee that an antigen or immunogen produced according to this sequence will have its desired biological activity.
In addition to developing a vaccine against leukemia-associated viruses, a reliable test for such leukemia-associated viruses is also needed. Generally, such tests have relied on the use of polyclonal antibodies raised to the whole or portions of a leukemia-associated virus. Unfortunately, the present tests not only give false positive results, but have also occasionally given false negative results. See Feline Leukema supra., pgs. 111 and 112. In addition, some of these tests rely on immunodiffusion and immunofluorescence which can be difficult to use as well as time consuming.
Accordingly, it would be desirable to develop an immunogen that could be used as part of a vaccine for the treatment and prevention of infections caused by a leukemia-associated virus. Such an immunogen would also be useful for raising antibodies that can either be used for the treatment of leukemia-associated virus diseases or as part of an assay system for such diseases. Such an assay system should be easy to use and reliable. The present invention meets these desires.
______________________________________ ABBREVIATIONS Three-Letter One-letter Amino Acid Abbreviation Symbol ______________________________________ Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Asparagine or Asx B aspartic acid Cysteine Cys C Glutamine Gln Q Glutamic acid Glu E Glutamine or glutamic acid Glx Z Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Adult T-Cell Leukemia Virus ATLV AKR Ecotropic Virus Akv Bovine Serum Albumin BSA Bovine Lacto Transfer BLOTTO Technique Optimizer Enzyme Multiplied Immunoassay (EMIT) Technique Enzyme-linked Immunosorbent (ELISA) Assay Feline Leukemia Virus FeLV Complete Freund's adjuvant CFA Incomplete Freund's adjuvant IFA Human T-Cell Leukemia Virus HTLV Keyhole Limpet Hemocyanin KLH Long Terminal Repeat LTR Mink Cell Focus-Forming Virus MCF Minimum Eagels Medium MEM Murine M Leukemia Virus MuLV Phosphate-buffered saline PBS Radioimmune assay RIA ______________________________________